Structure of GDAP1 bound to a product of lipid peroxidation

总结。

Summary. Mutations in GDAP1 are associated with the peripheral neuropathy Charcot-Marie-Tooth disease. Charcot-Ma- rie-Tooth is one of most common inherited neurological disorders, estimated to affect 126,000 people in the United States alone. GDAP1 knockdown, overexpression, and as well as multiple models of CMT show changes consistent with dysregulation of the cellular response to oxidative stress. These include changes in NRF2-driven transcriptional activity, evidence of glutathione depletion, redox disbalance, and mitochondrial depolarization. At the same time key aspects of the mitochondrial network’s response to oxidative stress are very similar to key aspects of CMT phenotypes: fragmentation, fusion deficits and change in position inside the cell. Finally, GDAP1 is a member of the Glutathione S-transferase (GST) superfamily which protect the cell against peroxidated lipids and xenobiotics, toxic molecules that accumulate under conditions of oxidative stress. The mechanism underly- ing GDAP1’s role in oxidative stress response is unknown. We have recently discovered that GDAP1 can bind 4-hydroxynoneal (4HNE), a toxic lipid that is formed from the breakdown of peroxidated lipids primarily in the mitochondria. This proposal will address two main questions: can GDAP1 utilize 4HNE as a substrate, in a manner similar to canonical GST enzymes, or does it have a non-enzymatic role in the oxidative stress re- sponse? Secondly, are there conformational changes associated with or a consequence of 4HNE binding? We will address these questions by 1) biochemically measuring enzymatic parameters associated with 4HNE medi- ated GST activity; 2) biochemically defining the impact of important residues within the putative active site pocket on 4HNE binding affinity and GST enzymatic activity; 3) determining the structure of the GDAP1-4HNE complex using x-ray crystallography. These data will define the molecular mechanism by which GDAP1 recognizes 4HNE to facilitate binding and reveal and conformational changes in protein that are associated with 4HNE binding. If GDAP1 is an enzyme it will reveal how it facilitates catalysis of 4HNE with glutathione. If GDAP1 is playing a non-enzymatic role, conformational changes resulting from 4HNE will play a key role in GDAP1 function and will be revealed in this structure. Overall, these studies will be critical in shaping future biochemical and cell-based investigations centered on GDAP1 function. Further, the structure will provide the foundation needed to compu- tationally predict small molecule binding partners, critical tools for modulating GDAP1 function that will allow deeper interrogation of CMT disease models and a first step towards therapeutic intervention.